In general, winterization is the name given to the process of removing sediment that appears in vegetable oils at low temperature. It originated from the early practice of allowing cottonseed oil to remain in outdoor storage during the cool winter months and filtering off the sediment-free oil. Dry fractional crystallization is a process wherein triglycerides with the highest melting temperature preferentially crystallize during cooling from a neat liquid (e.g., liquid lipid). After crystallization is complete, the solid phase is separated from the liquid phase by one of several types of physical processes. Alternatively, solvent crystallization is used to promote triglyceride crystal formation, because triglycerides at low temperature generally form more stable crystals with solvent than without solvent.
Docosahexaenoic acid (DHA)-rich lipid was extracted using conventional techniques and solvents (e.g., hexane) from Schizochytrium sp. biomass produced by fermentation, and the resulting extracted lipid, was winterized by chilling it to −2 to 2° C. followed by centrifugation. The lipid was then refined, bleached and deodorized, and put into gelatin capsules for sale as nutritional supplements. A problem arose with this product in that a haze would form in the product over time.
In one process for recovering lipids from biomass, as illustrated in FIG. 1, dried microalgae are suspended in commercial-grade n-hexane and wet milled. Hexane primarily extracts triglycerides, diglycerides, monoglycerides and esterified sterols, although other components of the total lipid fraction, such as phospholipids, free sterols and carotenoids, can also be extracted to a lesser degree. Centrifugation is employed to separate spent biomass from a lipid-rich miscella. The resultant mixture of lipid and solvent is referred to as miscella. The lipid content of the clarified miscella is adjusted to about 45 wt % using n-hexane. The miscella is winterized, in particular, the miscella is chilled to approximately −1° C., and held for 8 to 12 hours, to crystallize any saturated fats, or high melting point components. The miscella is then filtered to remove the crystallized stearin phase. Hexane is removed from the miscella, leaving behind the winterized lipid.
As illustrated in FIG. 2, the winterized lipid is heated and treated with citric acid or phosphoric acid to hydrate any phosphatides present in the lipid. Sodium hydroxide is added to neutralize any free fatty acids present. The resulting gums (hydrated phosphatides) and soapstock (neutralized fatty acids) are removed using a centrifuge. The lipid is mixed with water and re-centrifuged to remove any residual gum/soapstock. This step can be carried out with the first centrifugation. The refined lipid is bleached with silica and bleaching clay following pre-treatment with citric acid, to remove peroxides, color compounds, and traces of soapstock, phospholipids and metals. Filter aid is added at the end of the cycle to facilitate removal of the spent bleaching compounds from the lipid via filtration.
An additional step can be performed, where the bleached lipid is chilled to from about 5° C. to about 15° C. and held for about 6 to about 8 hours to crystallize any remaining stearines or waxes, if it is apparent that a sediment layer will form upon standing. Filter aid can be used to facilitate removal of the crystals via filtration, if this step is performed.
A deodorizer, operated at elevated temperatures under high vacuum, is used to destroy peroxides, which if left intact could later decompose and initiate free radical reactions. This step also removes any remaining low molecular weight compounds that can cause off-odors and flavors. Contact times in the deodorizer are minimized to prevent the formation of trans-fatty acids. Safe and suitable food approved antioxidants are added. The stabilized lipid is packaged in a phenolic-lined metal container under a nitrogen atmosphere to prevent oxidation.
The haze that formed in the lipid-filled gelatin capsules was analyzed and found to be composed of crystals of triglycerides containing myristic (14:0) and palmitic (16:0) fatty acids, a trisaturated fatty acid glyceride. These crystals had a melting point of about 50-55° C. The trisaturated glycerides comprised 6-8% of the crude extracted lipid. The above-described winterization process lowered the concentration of these trisaturated glycerides to <1%; however, not low enough to completely eliminate haze formation in the lipid. Additionally, about 30% of the lipids, and a corresponding 30% of the DHA, is removed in this traditional hexane (55% hexane and 45% crude oil) winterization process. Another problem was that when the temperature was lowered to crystallize the remaining <1% of the trisaturated triglycerides, more of the desired LCPUFA, e.g., disaturated triglycerides containing one DHA molecule, would also crystallize out. This would cause significant losses of the target product, DHA. Losses could be an additional 8-10% of the lipids. So by trying to solve one problem, another was created. It would be desirable to have a process by which the LCPUFA level could be maintained at a desirably high level and the haze could be reduced or eliminated.